Transgenic plants containing ligninase and cellulase which degrade lignin and cellulose to fermentable sugars

ABSTRACT

The present invention provides transgenic plants which after harvest degrade the lignin and cellulose therein to fermentable sugars which can further be fermented to ethanol or other products. In particular, the transgenic plants comprise ligninase and cellulase genes from microbes operably linked to a DNA encoding a signal peptide which targets the fusion polypeptide produced therefrom to an organelle of the plant, in particular the chloroplasts. When the transgenic plants are harvested, the plants are ground to release the ligninase and cellulase which then degrade the lignin and cellulose of the transgenic plants to produce the fermentable sugars.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application 09/981,900 filed Oct.18, 2001, now U.S. Pat. No. 7,049,485 which claims priority toprovisional application Ser. No. 60/242,408, filed Oct. 20, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

Reference to a “Nucleotide/Amino Acid Sequence Listing AppendixSubmitted on a Compact Disc”

The application contains nucleotide and amino acid sequences which areidentified with SEQ ID NOs. A compact disc is provided which containsthe Sequence Listings for the sequences. The Sequence Listing on thecompact disc and is identical to the paper copy of the Sequence Listingprovided with the application.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to transgenic plants which after harvestdegrade the lignin and cellulose therein to fermentable sugars which canfurther be fermented to ethanol or other products. In particular, thetransgenic plants comprise ligninase and cellulase genes from microbesoperably linked to a DNA encoding a signal peptide which targets thefusion polypeptide produced therefrom to an organelle of the plant, inparticular the chloroplasts. When the transgenic plants are harvested,the plants are ground to release the ligninase and cellulase which thendegrade the lignin and cellulose of the transgenic plants to produce thefermentable sugars.

(2) Description of Related Art

If human economies are to become more sustainable, then it is imperativethat humans learn how to use the solar energy that is carbon-fixed inplant biomass to meet a larger fraction of energy and raw materialneeds. About 180 billion tons of new plant matter (biomass) is producedannually worldwide. Thus, about 30 tons of plant matter per person isproduced every year. In North America, about three tons of plant matteris used per person every year. That means that the energy value ofnaturally produced biomass is equivalent to ten times the total humanuse of all types of energy. However, because of the difficulty inextracting the energy from plant biomass, most of the energy potentialof the biomass goes unused.

At present, the United States produces ethanol from starch produced incorn grain using amylase enzymes to degrade the starch to fermentablesugars. Much of the ethanol that is produced from corn grain is exportedto Brazil where it is efficiently used to power transportation vehicles.In general, while the corn grain is used in the production of ethanol,the remainder of the corn biomass, i.e., the leaves and stalks, isseldom unused because of the cost in degrading the leaves and stalkscomprising lignins and cellulose, generally in the form oflignocellulose, to fermentable sugars. The lignocellulose in the stalksand leaves of corn biomass represents a tremendous source of untappedenergy that goes unused because of the difficulty and cost of convertingit to fermentable sugars.

Currently, there are four technologies available to convert cellulose tofermentable sugars. These are concentrated acid hydrolysis, dilute acidhydrolysis, biomass gasification and fermentation, and enzymatichydrolysis.

Concentrated acid hydrolysis is based on concentrated acidde-crystallization of cellulose followed by dilute acid hydrolysis tosugars at near theoretical yields. Separation of acid from sugars, acidrecovery, and acid re-concentration are critical unit operations. Theconcentrated sulfuric acid process has been commercialized in the past,particularly in the former Soviet Union, Germany, and Japan. However,these processes were only successful during times of national crisis,when economic competitiveness of ethanol production could be ignored.

Dilute acid hydrolysis occurs in two stages to maximize sugar yieldsfrom the hemicellulose and cellulose fractions of biomass. The firststage is operated under milder conditions to hydrolyze hemicellulose,while the second stage is optimized to hydrolyze the more resistantcellulose fraction. Liquid hydrolyzates are recovered from each stage,neutralized, and fermented to ethanol. As indicated earlier, Germany,Japan, and Russia have operated dilute acid hydrolysis percolationplants off and on over the past 50 years. However, the technologyremains non-competitive for the conversion of cellulose to fermentablesugars for production of ethanol.

In biomass gasification and fermentation, biomass is converted to asynthesis gas, which consists primarily of carbon monoxide, carbondioxide, and hydrogen) via a high temperature gasification process.Anaerobic bacteria are then used to convert the synthesis gas intoethanol.

In early processes embracing enzymatic hydrolysis of biomass to ethanol,the acid hydrolysis step was replaced with an enzyme hydrolysis step.This process scheme was often referred to as separate hydrolysis andfermentation (SHF) (Wilke et al., Biotechnol. Bioengin. 6: 155-175(1976)). In SHF, pretreatment of the biomass is required to make thecellulose more accessible to the enzymes. Many pretreatment options havebeen considered, including both thermal and chemical steps. The mostimportant process improvement made for the enzymatic hydrolysis ofbiomass was the introduction of simultaneous saccharification andfermentation (SSF) U.S. Pat. No. 3,990,944 to Gauss et al. and U.S. Pat.No. 3,990,945 to Huff et al.). This process scheme reduced the number ofreactors involved by eliminating the separate hydrolysis reactor and,more importantly, avoiding the problem of product inhibition associatedwith enzymes.

In the presence of glucose, β-glucosidase stops hydrolyzing cellobiose.The build up of cellobiose, in turn, shuts down cellulose degradation.In the SSF process scheme, cellulase enzyme and fermenting microbes arecombined. As sugars are produced by the enzymes, the fermentativeorganisms convert them to ethanol. The SSF process has, more recently,been improved to include the co-fermentation of multiple sugarsubstrates in a process known as simultaneous saccharification andco-fermentation (SSCF) (www.ott.doe.gov/biofuels/enzymatic.html).

While cellulase enzymes are already commercially available for a varietyof applications. Most of these applications do not involve extensivehydrolysis of cellulose. For example, the textile industry applicationsfor cellulases require less than 1% hydrolysis. Ethanol production, bycontrast, requires nearly complete hydrolysis. In addition, most of thecommercial applications for cellulase enzymes represent higher valuemarkets than the fuel market. For these reasons, enzymatic hydrolysis ofbiomass to ethanol remains non-competitive.

However, while the above processes have focused on converting celluloseto fermentable sugars or other products, much of the cellulose in plantbiomass is in the form of lignocellulose. Lignin is a complexmacromolecule consisting of aromatic units with several types ofinter-unit linkages. In the plant, the lignin physically protects thecellulose polysaccharides in complexes called lignocellulose. To degradethe cellulose in the lignocellulose complexes, the lignin must first bedegraded. While lignin can be removed in chemi-mechanical processes thatfree the cellulose for subsequent conversion to fermentable sugars, thechemi-mechanical processes are inefficient. Ligninase and cellulaseenzymes, which are produced by various microorganisms, have been used toconvert the lignins and cellulose, respectively, in plant biomass tofermentable sugars. However, the cost for these enzymes is expensive,about six dollars a pound. As long as the cost to degrade plant biomassremains expensive, the energy locked up in the plant biomass willlargely remain unused.

An attractive means for reducing the cost of degrading plant biomass isto make transgenic plants that contain cellulases. For example, WO98/11235 to Lebel et al. discloses transgenic plants that expresscellulases in the chloroplasts of the transgenic plants or transgenicplants wherein the cellulases are targeted to the chloroplasts.Preferably, the cellulases are operably linked to a chemically-induciblepromoter to restrict expression of the cellulase to an appropriate time.However, because a substantial portion of the cellulose in plants is inthe form of lignocellulose, extracts from the transgenic plants areinefficient at degrading the cellulose in the lignocellulose.

U.S. Pat. No. 5,981,835 to Austin-Phillips et. al. discloses transgenictobacco and alfalfa which express the cellulases E2, or E3 fromThermomononospora fusca. The genes encoding the E2 or E3, which weremodified to remove their leader sequence, were placed under the controlof a constitutive promoter and stably integrated into the plant genome.Because the leader sequence had been removed, the E2 or E3 productpreferentially accumulated in the cytoplasm of the transgenic plants.However, because the cellulase can leak out of the cytoplasm and intothe cell wall where it can degrade cellulose in the cell wall, thegrowth of the transgenic plants can be impaired.

U.S. Pat. No. 6,013,860 to Himmel et al. discloses transgenic plantswhich express the cellulase E1 from Acidothermus cellulolyticus. Thegene encoding E1, which was modified to remove the leader region, wasplaced under the control of a plastid specific promoter and preferablyintegrated into the plastid genome. Because the leader sequence had beenremoved, the E1 product accumulated in the plastid.

While the above transgenic plants are an improvement, accumulation ofcellulytic enzymes in the cytoplasm of a plant is undesirable sincethere is the risk that the cellulase can leak out from the cytoplasm andinjure the plant. For example, research has shown that plants such asthe avocado, bean, pepper, peach, poplar, and orange also containcellulase genes, which are activated by ethylene during ripening andleaf and fruit abscission. Therefore, transgenic plants which containlarge quantities of cellulase in the cytoplasm are particularly prone todamage. Furthermore, the cellulases accumulate in all tissues of theplant which can be undesirable. Restriction of cellulase expression toplastids is desirable because it reduces the risk of plant damage duethe cellulases leaking from the cell. However, for most crop plants, ithas been difficult to develop a satisfactory method for introducingheterologous genes into the genome of plastids. Furthermore, cellulaseis expressed in all tissues which contain plastids which can beundesirable.

For production of ligninases to use in degrading lignins, the ligninasesof choice are from the white-rot fungus Phanerochaete chrysosporium. Oneof the major lignin-degrading, extracellular enzymes produced by P.chrysosporium is lignin peroxidase (LIP). Potential applications of LIPinclude not only lignin degradation but also biopulping of wood andbiodegradation of toxic environmental pollutants. To produce largequantities of LIP, the fungus can be grown in large reactors and theenzyme isolated from the extracellular fluids. However, the yields havebeen low and the process has not been cost-effective. Production ofrecombinant LIP in E. coli, in the fungus Trichoderma reesei, andbaculovirus have been largely unsuccessful. Heterologous expression oflignin-degrading manganese peroxidase in alfalfa plants has beenreported; however, the transgenic plants had reduced growth andexpression of the enzyme was poor (Austin et al., Euphytica 85: 381-393(1995)).

Although difficult to sufficiently and cheaply produce ligninases innon-plant systems, ligninases have evoked worldwide interest because oftheir potential in degrading a variety of toxic xenobiotic compoundssuch as PCBs and benzo(a)pyrenes in the environment (Yadav et al., Appl.Environ. Microbiol. 61: 2560-2565 (1995); Reddy, Curr. Opin. Biotechnol.6: 320-328 (1995); Yadav et al., Appl. Environ. Microbiol. 61: 677-680(1994)).

Therefore, a need remains for an economical method for making transgeniccrop plants wherein the ligninase and cellulase genes are incorporatedinto the plant genome but wherein the ligninase and cellulase expressionare restricted to particular plant tissues, e.g., the leaves, and theligninase and cellulase products are directed to a plant organellewherein it accumulates without damaging the transgenic plant.

SUMMARY OF THE INVENTION

The present invention provides transgenic plants which after harvestdegrade the lignin and cellulose therein to fermentable sugars which canfurther be fermented to ethanol or other products. In particular, thetransgenic plants comprise ligninase and cellulase genes from microbesoperably linked to a DNA encoding a signal peptide which targets thefusion polypeptide produced therefrom to an organelle of the plant, inparticular the chloroplasts. When the transgenic plants are harvested,the plants are ground to release the ligninase and cellulase which thendegrade the lignin and cellulose of the transgenic plants to produce thefermentable sugars.

Therefore, the present invention provides a transgenic plant whichdegrades lignocellulose when the transgenic plant is ground to produce aplant material comprising (a) at least one DNA encoding a cellulasewhich is operably linked to a nucleotide sequence encoding a signalpeptide wherein the signal peptide directs the cellulase to an organelleof the transgenic plant; and (b) at least one DNA encoding a ligninasewhich is operably linked to a nucleotide sequence encoding a signalpeptide wherein the signal peptide directs the ligninase to theorganelle of the transgenic plant, wherein the transgenic plant degradesthe lignocellulose when ground to produce the plant material.

Further, the present invention provides a transgenic plant whichdegrades lignins when the transgenic plant is ground to produce a plantmaterial comprising at least one DNA encoding a ligninase which isoperably linked to a nucleotide sequence encoding a signal peptidewherein the signal peptide directs the ligninase to an organelle of thetransgenic plant wherein the transgenic plant degrades the lignins whenground to produce the plant material.

Further still, the present invention provides a transgenic plant whichdegrades cellulose when the transgenic plant is ground to produce aplant material comprising at least one DNA encoding a cellulase which isoperably linked to a nucleotide sequence encoding a signal peptidewherein the signal peptide directs the cellulase to an organelle of thetransgenic plant wherein the transgenic plant degrades the cellulosewhen ground to produce the plant material.

The present invention also provides a method for producing a transgenicplant which degrades lignocellulose when the transgenic plant is groundto produce a plant material.

In one embodiment, the method comprises (a) providing a first transgenicplant which includes a DNA encoding a cellulase which is operably linkedto a nucleotide sequence encoding a signal peptide wherein the signalpeptide directs the cellulase to an organelle of the transgenic plantand a second transgenic plant which includes a DNA encoding a ligninasewhich is operably linked to a nucleotide sequence encoding a signalpeptide wherein the signal peptide directs the ligninase to theorganelle of the transgenic plant; and (b) mating by sexualfertilization the first and the second transgenic plants to produce athird transgenic plant which includes the first DNA encoding thecellulase and the second DNA encoding the ligninase, wherein thetransgenic plant degrades the lignocellulose when ground to produce theplant material.

In a further embodiment of the above method, the progeny of the thirdtransgenic plant are mated by sexual fertilization to a transgenic plantselected from the group consisting of the first, second, and thirdtransgenic plants to produce a transgenic plant comprising multiples ofgenes encoding cellulases and ligninases.

The present invention further provides a method for convertinglignocellulose in a plant material to fermentable sugars.

In one embodiment, the method comprises (a) providing a transgenic plantwhich includes at least one DNA encoding a cellulase which is operablylinked to a nucleotide sequence encoding a signal peptide wherein thesignal peptide directs the cellulase to an organelle of the transgenicplant and at least one DNA encoding a ligninase which is operably linkedto a nucleotide sequence encoding a signal peptide wherein the signalpeptide directs the ligninase to the organelle of the transgenic plant;(b) growing the transgenic plant for a time sufficient for thetransgenic plant to accumulate a sufficient amount of the cellulase andthe ligninase in the organelle of the transgenic plant; (c) harvestingthe transgenic plant which has accumulated the cellulase and ligninasein the organelle of the transgenic plant; (d) grinding the transgenicplant for a time sufficient to produce the plant material wherein thecellulase and ligninase produced by the transgenic plant are releasedfrom the organelle of the transgenic plant; (e) incubating the plantmaterial for a time sufficient for the cellulase and ligninase in theplant material to produce the fermentable sugars from the lignocellulosein the plant material; and (f) extracting the fermentable sugarsproduced from the lignocellulose by the cellulase and the ligninase fromthe plant material.

In another embodiment, the method comprises (a) providing a transgenicplant which includes at least one DNA encoding a cellulase which isoperably linked to a nucleotide sequence encoding a signal peptidewherein the signal peptide directs the cellulase to an organelle of thetransgenic plant; (b) growing the transgenic plant for a time sufficientfor the transgenic plant to accumulate a sufficient amount of thecellulase in the organelle of the transgenic plant; (c) harvesting thetransgenic plant which has accumulated the cellulase in the organelle ofthe transgenic plant; (d) grinding the transgenic plant for a timesufficient to produce a plant material wherein the cellulase is releasedfrom the organelle in the transgenic plant; (e) mixing the plantmaterial with a fungus that produces a ligninase; (f) incubating thetransgenic plant material with the fungus for a time sufficient for thecellulase released from the transgenic plant and the ligninase providedby the fungus to degrade the lignocellulose in the plant material toproduce the fermentable sugars; and (g) extracting the fermentablesugars produced from the lignocellulose in the plant material.

In a further embodiment of either one of the above embodiments of themethod for converting lignocellulose in a plant material to fermentablesugars, the plant material further includes a plant material made from anon-transgenic plant.

In a further still embodiment of the either one of the above methods,the fermentable sugars are fermented to ethanol.

In a further embodiment of the transgenic plant for any one of theaforementioned embodiments of the present invention wherein thetransgenic plant expresses cellulase and ligninase or the cellulasewithout the ligninase, the DNA encoding the cellulase is from anorganism selected from the group consisting of Trichoderma reesei,Acidothermus cellulyticus, Streptococcus salivarius, Actinomycesnaeslundi, and Thermomonospora fusca.

In a further still embodiment of the above transgenic plant, the DNAencoding the cellulase is selected from the group consisting of an e1gene from Acidothermus cellulyticus, a cbh1 gene from Trichodermareesei, a dextranase gene from Streptococcus salivarius, and abeta-glucosidase gene from Actinomyces naeslundi.

In a further still embodiment of the above transgenic plant, the e1 genecomprises the nucleotide sequence set forth in SEQ ID NO:4, the cbh1gene comprises the nucleotide sequence set forth in SEQ ID NO:10, thedextranase gene comprises the nucleotide sequence set forth in SEQ IDNO:8, and the beta-glucosidase gene comprises the nucleotide sequenceset forth in SEQ ID NO:6.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention wherein the transgenic plantexpresses cellulase and ligninase or the ligninase without thecellulase, the DNA encoding the ligninase is from Phanerochaetechrysosporium.

In a further still embodiment of the above transgenic plant, theligninase is ckg4 comprising the nucleotide sequence set forth in SEQ IDNO:11 or ckg5 comprising the nucleotide sequence set forth in SEQ IDNO:13.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention, the DNA encoding thecellulase and the DNA encoding the ligninase are each operably linked toa leaf-specific promoter. In a particular embodiment, the leaf-specificpromoter is a promoter for rbcS.

In a further still embodiment of the above transgenic plant, thenucleotide sequence encoding the signal peptide encodes a signal peptideof rbcS.

In a further still embodiment of the above transgenic plant, the rbcScomprises the nucleotide sequence set forth in SEQ ID NO:1.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention, the transgenic plant isselected from the group consisting of maize, wheat, barley, rye, hops,hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco,alfalfa, arabidopsis, coniferous tree, and deciduous tree.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention, the DNAs encoding thecellulase, ligninase, or both are stably integrated into nuclear orplastid DNA of the transgenic plant.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention, the transgenic plant furtherincludes a DNA encoding a selectable marker operably linked to aconstitutive promoter.

In a further still embodiment of the above transgenic plant, the DNAencoding the selectable marker provides the transgenic plant withresistance to an antibiotic, an herbicide, or to environmental stress.

In a further still embodiment of the above transgenic plant, the DNAencoding resistance to the herbicide is a DNA encoding phosphinothricinacetyl transferase which confers resistance to the herbicidephosphinothricin.

In a further still embodiment for any one of the aforementionedtransgenic plants of the present invention, the organelle of thetransgenic plant is selected from the group consisting of nucleus,microbody, endoplasmic reticulum, endosome, vacuole, mitochondria,chloroplast, or plastid.

In a further still embodiment of the above transgenic plant, theorganelle of the transgenic plant is the chloroplast.

In a further still embodiment of the transgenic plants which degradelignocellulose or cellulose, the lignocellulose is degraded tofermentable sugars which can then be fermented to ethanol.

OBJECTS

It is an object of the present invention to provide transgenic plantswhich degrade lignocellulose to fermentable sugars, methods for makingthe transgenic plants which degrade lignocellulose, and methods forusing the transgenic plants to degrade lignocellulose to fermentablesugars.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a plasmid containing a heterologous geneexpression cassette containing cbh1 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a heterologous geneexpression cassette containing the bar gene operably linked to the Act1promoter. rbcSP is the rbcS gene promoter, SP is DNA encoding the rbcSsignal peptide, pin3′ is the 3′ untranslated region of the potatoinhibitor II-chloramphenicol acetyltransferase gene, Act1 is thepromoter for the act1 gene, and nos is the 3′ untranslated region of theAgrobacterium nopaline synthase gene.

FIG. 2 is a diagram of a plasmid containing a heterologous geneexpression cassette containing e1 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a heterologous geneexpression cassette containing the bar gene operably linked to the Act1promoter. The terms in the diagram are as in FIG. 1.

FIG. 3 is a diagram of a heterologous gene expression cassettecontaining the bar gene in plasmid pDM302. Act1 is the promoter for theact1 gene and nos is the 3′ untranslated region of the Agrobacteriumnopaline synthase gene.

FIG. 4 is a diagram of plasmid pSMF13 which is plasmid pSK containing aheterologous gene expression cassette containing cbh1 operably linked tothe rbcS promoter. The terms in the diagram are as in FIG. 1.

FIG. 5 is a diagram of plasmid pMSF14 which is plasmid pSK containing aheterologous gene expression cassette containing cbh1 operably linked tothe rbcS promoter and DNA encoding the rbcS signal peptide. The terms inthe diagram are as in FIG. 1.

FIG. 6 is a diagram of plasmid pMSF15 which is plasmid pBI221 containinga heterologous gene expression cassette containing syn-cbh1 operablylinked to the rbcS promoter and DNA encoding the rbcS signal peptide.The terms in the diagram are as in FIG. 1.

FIG. 7 is a diagram of plasmid pTZA8 which is plasmid pBI121 containinga heterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter and DNA encoding the SSU signal peptide. SSU is theglycine max (soybean) rbcS signal peptide. CaMV35S is the cauliflowermosaic virus 35S promoter. The remainder of the terms are as in thediagram are as in FIG. 1.

FIG. 8 is a diagram of plasmid pZA9 which is plasmid pBI121 containing aheterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter and DNA encoding the VSP signal peptide. VSP is thesoybean vegetative storage protein beta-leader sequences. The remainderof the terms in the diagram are as in FIG. 7.

FIG. 9 is a diagram of plasmid pZA10 which is plasmid pBI121 containinga heterologous gene expression cassette containing e1 operably linked tothe CaMV35S promoter. The remainder of the terms in the diagram are asin FIG. 7.

FIG. 10 is a diagram of a plasmid containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a gene expression cassettecontaining the bar gene operably linked to the Act1 promoter. Theremainder of the terms in the diagram are as in FIG. 1.

FIG. 11 is a diagram of a plasmid containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide and a gene expression cassettecontaining the bar gene operably linked to the Act1 promoter. Theremainder of the terms in the diagram are as in FIG. 1.

FIG. 12 is a diagram of plasmid pSMF18 containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcSpromoter. The remainder of the terms in the diagram are as in FIG. 1.

FIG. 13 is a diagram of plasmid pSMF19 containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcSpromoter. The remainder of the terms in the diagram are as in FIG. 1.

FIG. 14 is a diagram of plasmid pSMF16 containing a heterologous geneexpression cassette containing ckg4 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide. The remainder of the terms inthe diagram are as in FIG. 1.

FIG. 15 is a diagram of plasmid pSMF17 containing a heterologous geneexpression cassette containing ckg5 operably linked to the rbcS promoterand DNA encoding the rbcS signal peptide. The remainder of the terms inthe diagram are as in FIG. 1.

DESCRIPTION OF PREFERRED EMBODIMENTS

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The term “cellulase” is used herein as a generic term that includesendoglucanases such as the EI beta-1,4-endoglucanase precursor gene (e1)of Acidothermus cellulolyticus and exoglucanases such as thecellobiohydrolase gene (cbh1) of Trichoderma reesei (also classified bysome as Trichoderma longibrachiatum), the dextranase gene ofStreptococcus salivarius encoding the 1,6-alpha-glucanhydrolase gene,and the beta-glucosidase gene from Actinomyces naeslundi. Endoglucanasesrandomly cleave cellulose chains into smaller units. Exoglucanasesinclude cellobiohydrolases, which liberate glucose dimers (cellobiose)from the ends of cellulose chains; glucanhydrolases, which liberateglucose monomers from the ends of cellulose chains; and,beta-glucosidases, which liberate D-glucose from cellobiose dimers andsoluble cellodextrins. When all four of the above enzymes are combined,they work synergistically to rapidly decrystallize and hydrolyzecellulose to fermentable sugars.

The term “lignin” is used herein as a generic term that includes bothlignins and lignocelluloses.

The term “ligninase” is used herein as a generic term that includes allvarieties of enzymes which degrade lignins such as the lignin peroxidasegene of Phanerochaete chrysosporium.

A variety of fungi and bacteria produce ligninase and cellulase enzymes,and based on evolutionary pressures, these fungi are able to degradelignin or cellulose and hemicellulose of plant residues in the soil. Inthe laboratory, cellulases have been used to hydrolyze or convertcellulose and hemicellulose into mixtures of simple sugars that can beused in fermentation to produce a wide variety of useful chemical andfuel products, including but not limited to, ethanol, lactic acid, and1,3-propanediol, which is an important molecular building block in theproduction of environmentally-friendly plastics.

The biodegradation of lignin, which comprises 20-30% of the dry mass ofwoody plants, is of great economic importance because this process isbelieved to be an important rate-limiting step in the earth's carboncycle. Furthermore, there is considerable potential for thetransformation of lignin into aromatic chemical feedstock. Also,delignification of lignocellulosic feeds has been shown to increasetheir digestibility by cattle by about 30%, therefore, contributing toenhanced cost effectiveness for producing milk and meat. Moreover,research on lignin biodegradation has important implications inbiopulping and biobleaching in the paper industry.

The present invention provides transgenic plants which produceligninases, cellulases, or both in the leaves and straw/stalks of theplant. While the transgenic plant can be any plant which is practicalfor commercial production, it is preferable that the transgenic plantsbe constructed from plants which are produced in large quantities andwhich after processing produce a substantial amount of leaves and stalksas a byproduct. Therefore, it is desirable that the transgenic plant beconstructed from plants including, but not limited to, maize, wheat,barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane,clover, tobacco, alfalfa, arabidopsis, coniferous trees, and deciduoustrees. Most preferably, the transgenic plant is constructed from maize.

Maize is a preferred plant because it is a major crop in the UnitedStates; approximately 60 million acres of maize are produced per year.Further, there is already a large industry built around the processingof maize grain to industrial products, which includes the production ofover 1.2 billion gallons of fuel ethanol per year. Thus, fermentablesugars produced by the hydrolysis of maize stalks and leaves accordingto the present invention can be utilized within the large existing maizerefining infrastructure. Leaves and stalks from transgenic maize madeaccording to the present invention can be made available to thisrefining infrastructure in large quantities, about tens of millions oftons annually) at a current cost of about 30 dollars per ton. This costis about one quarter of the cost of maize grain which further enhancesthe value of the present invention for the economical production of awide variety of industrial products from the residue of transgenicplants made according to the present invention. Furthermore, maize ispreferred because it is a C-4 monocot that has very large chloroplasts.The large chloroplasts enables the chloroplasts of the transgenic maizeof the present invention to accumulate higher levels of ligninases andcellulases than could be accumulated in the chloroplasts of othertransgenic plants, e.g., C-3 dicots and monocots. Therefore, transgenicmaize of the present invention is a particularly useful source ofligninases and cellulases.

Thus, the transgenic plants of the present invention provide aplentiful, inexpensive source of fungal or bacterial ligninases andcellulases which can be used to degrade lignins and cellulose in plantsto fermentable sugars for production of ethanol or for other uses whichrequire ligninases and cellulases such as pre-treating silage toincrease the energy value of lignocellulosic feeds for cows and otherruminant animals, pre-treating lignocellulosic biomass for fermentativeconversion to fuels and industrial chemicals, and biodegradation ofchloroaromatic environmental pollutants. Because the transgenic plantsof the present invention produce the ligninases, cellulases, or boththerein, the external addition of ligninases and cellulases fordegradation of the plant material is no longer necessary. Therefore, thepresent invention enables the plant biomass, which is destined to becomeethanol or other products, to serve as the source of ligninase andcellulase. Furthermore, the plant material from the transgenic plants ofthe present invention can be mixed with non-transgenic plant material.The ligninases, cellulases, or both produced by the transgenic plantswill degrade the lignin and cellulose of all the plant material,including the non-transgenic plant material. Thus, ligninase andcellulase degradation of plant material can be carried out moreeconomically.

The transgenic plants of the present invention comprise one or moreheterologous gene expression cassettes containing DNA encoding at leastone fungal or bacterial ligninase, cellulase, or both inserted into theplant's nuclear genome. The preferred cellulase is encoded by a DNA fromthe microorganism Acidothermus cellulolyticus, Thermomonospora fusca,and Trichoderma reesei (Trichoderma longibrachiatum). Othermicroorganisms which produce cellulases suitable for the presentinvention include Zymomonas mobilis, Acidothermus cellulolyticus,Clostridium thermocellum, Eiwinia chrysanthemi, Xanthomonas campestris,Alkalophilic Bacillus sp., Cellulomonas fimi, wheat straw mushroom(Agaricus bisporus), Ruminococcus flavefaciens, Ruminococcus albus,Fibrobacter succinogenes, and Butyrivibrio fibrisolvens.

The preferred ligninase is lignin peroxidase (LIP) encoded in DNA fromPhanerochaete chrysosporium or Phlebia radiata. One of the majorlignin-degrading, extracellular enzymes produced by P. chrysosporium isLIP. The LIPs are glycosylated heme proteins (MW 38 to 46 kDa) which aredependent on hydrogen peroxide for activity and catalyze the oxidativecleavage of lignin polymer. At least six heme proteins (H1, H2, H6, H7,H8, and H10) with LIP activity have been identified in P. chrysosporiumstrain BKMF-1767 of which isozymes H2, H6, H8, and H10 are the majorLIPs in both static and agitated cultures of P. chrysosporium. However,other fungi which produce ligninases suitable for use in the presentinvention include Bjerkandera adusta, Trametes hirsuta, Plebia radiata,Pleurotus spp., Stropharia aurantiaca, Hypholoma fasciculare, Trametesversicolor, Gymnopilus penetrnas, Stereum hirsutum, Mycena haematopus,and Armillaria mellea.

In the present invention, the transgenic plant comprises a DNA encodingone or more cellulase fusion proteins wherein the DNA encoding thecellulases are operably linked to a DNA encoding a signal peptide whichdirects the cellulase fusion protein to a plant organelle such as thenucleus, a microbody (e.g., a peroxisome, or specialized versionthereof, such as a glyoxysome), an endoplasmic reticulum, an endosome, avacuole, a mitochondria, a chloroplast, or a plastid. By sequesteringthe cellulase fusion proteins in the plant organelle, the cellulasefusion protein is prevented from leaking outside the cytoplasm to harmthe plant by degrading the cellulose in the plant's cell wall while theplant is being cultivated. In particular embodiments of the presentinvention, the gene encoding the cellulase is modified by replacing theamino acid codons that encode the leader region of the cellulase withamino acid codons that encode the signal peptide.

In a preferred embodiment of the invention, the amino acid codons thatencode the signal peptide that directs the protein to which it isattached to the plant organelle, the chloroplasts, are the nucleotidecodons that encode the rice rubisco synthase gene (rbcS) small subunitsignal peptide (rbcSSP). The nucleotide sequence of the rbcS is setforth in SEQ ID NO:1 (GenBank Accession No. X07515). The 47 amino acidsignal peptide of the rbcS protein has the amino acid sequence MAPPSVMASS ATIVA PFQGS SPPPA CRRPP SELQL RQRQH GGRIR CM (SEQ ID NO:2). TherbcS SP directs proteins to which it is operably linked to thechloroplasts of the transgenic plant. Therefore, in the preferredembodiment of the present invention, the transgenic plant comprises aDNA encoding the cellulase operably linked with a DNA encoding the rbcSSP to produce the cellulase fusion protein. The rbcS SP directs thecellulase fusion protein to the chloroplasts. Thus, the cellulase fusionprotein produced by the transgenic plant accumulates in the chloroplastsof the transgenic plant which protects the transgenic plant fromdegradation by the cellulase fusion protein while it is beingcultivated. Alternatively, the DNA encoding the cellulase is modified atits 3′ end to encode a transit peptide such as the peptide RAVARL (SEQID NO:3), which targets the ligninase fusion protein to the peroxisomes(U.S. Pat. No. 6,103,956 to Srienc et al.). Preferably, the leaderregion of the cellulase is also removed. In any one of the aboveembodiments, the cellulase can be further modified to include a GCcontent that approximates the GC content of the genomic DNA of the plantby methods well known in the art.

In a preferred embodiment, the cellulase comprising the cellulase fusionprotein is encoded by the EI beta-1,4-endoglucanase precursor gene (e1)of Acidothermus cellulolyticus, the cellobiohydrolase gene (cbh1) ofTrichoderma reesei (Trichoderma longibrachiatum), the beta-glucosidasegene from Actinomyces naeslundi, or the glucanhydrolase (dextranase)gene from Streptococcus salivarius. The nucleotide sequence of the e1DNA is set forth in SEQ ID NO:4 (GenBank Accession No. U33212), whichencodes the cellulase with the amino acid sequence set forth in SEQ IDNO:5. SEQ ID NO:6 provides the nucleotide sequence of thebeta-glucosidase gene from Actinomyces naeslundi (GenBank Accession No.AY029505), which encodes the beta-glucosidase with the amino acidsequence set forth in SEQ ID NO:7. SEQ ID NO:8 provides the nucleotidesequence of the dextranase gene from Streptococcus salivarius (GenBankAccession No. D29644), which encodes a glucanhydrolase with the aminoacid sequence set forth in SEQ ID NO:9. The nucleotide sequence of cbh1is set forth in SEQ ID NO:10 (GenBank Accession No. E00389), whichencodes the cellulase that includes the joined exons from positions 210to 261, 738 to 1434, and 1498-1881.

In the present invention, the transgenic plant comprises a DNA encodingone or more ligninase fusion proteins wherein a DNA encoding theligninase is operably linked to a DNA encoding a signal peptide whichdirects the ligninase fusion protein to a plant organelle. Bysequestering the ligninase fusion proteins in the plant organelles, themodified ligninase is prevented from leaking outside the cytoplasm toharm the plant by degrading the ligninase in the plant's cell wall whilethe plant is being cultivated. In particular embodiments of the presentinvention, the leader sequence of the gene encoding the ligninase ismodified by replacing the amino acid codons that encode the leaderregion of the ligninase with amino acid codons that encode the signalpeptide.

In a preferred embodiment of the invention, the amino acid codons thatencode the signal peptide are the amino acid codons which encode therice rubisco synthase gene (rbcS) small subunit signal peptide (rbcSSP).The nucleotide sequence of the rbcS is set forth in SEQ ID NO:1 (GenBankAccession No. X07515). The 47 amino acid signal peptide of the rbcSprotein has the amino acid sequence MAPPS VMASS ATIVA PFQGS SPPPA CRRPPSELQL RQRQH GGRIR CM (SEQ ID NO:2). Therefore, in the preferredembodiment of the present invention, the transgenic plant comprises aDNA encoding the ligninase operably linked to a DNA encoding the rbcSSP. The rbcS SP directs the ligninase fusion protein to thechloroplasts. Thus, the ligninase fusion protein produced by thetransgenic plant accumulates in the chloroplasts of the transgenic plantwhich protects the transgenic plant from degradation by the ligninasefusion protein while it is being cultivated. Alternatively, the DNAencoding the ligninase is modified at its 3′ end to encode a transitpeptide such as the peptide RAVARL (SEQ ID NO:3). Optionally, the leaderregion of the ligninase is also removed. In any one of the aboveembodiments, the ligninase can be further modified to include a GCcontent that approximates the GC content of the genomic DNA of the plantby methods well known in the art.

In a preferred embodiment of the invention, the ligninase comprising theligninase fusion protein is encoded by the lignin peroxidase gene (LIP)genes ckg4 (H2) and ckg5 (H10) of Phanerochaete crysosporium (de Boer etal., Gene 6: 93-102 (1987), Corrigendum in Gene 69: 369 (1988)). Thenucleotide sequence of the ckg4 gene is set forth in SEQ ID NO:11(GenBank Accession No. M18743), which encodes the amino acid with thesequence set forth in SEQ ID NO:12. The nucleotide sequence of the ckg5gene is set forth in SEQ ID NO:13 (GenBank Accession No. M18794), whichencodes the amino acid with the sequence set forth in SEQ ID NO:14.

In the present invention, transcription and, therefore, expression ofthe ligninase and cellulase fusion proteins are effected by a promoterthat is active in a particular tissue of the plant, e.g., a promoterthat is active primarily in the leaves of a plant. A leaf-specificpromoter that is preferred for transcription (expression at the RNAlevel) is the rice rubisco synthase gene promoter (rbcSP), which has thenucleotide sequence prior to the rbcS gene coding region included in SEQID NO:1. In some embodiments of the present invention, it is desirableto relegate transcription of the heterologous gene expression cassetteto the seeds using a seed-specific promoter. Seed-specific promotersthat are suitable include, but are not limited to, the seed-specificpromoters such as the maize 19 kDa zein (cZ19B1) promoter, the maizecytokinin-induced message (Cim1) promoter, and the maizemyo-inositol-1-phosphate synthase (mi1ps) promoter, which are disclosedin U.S. Pat. No. 6,225,529 to Lappegard et al. Therefore, in theheterologous gene expression cassettes, the nucleotide sequencecomprising rbcS promoters are operably linked to the nucleotidesequences encoding the ligninase and cellulase fusion proteins. Thus, ina transgenic plant of the present invention, transcription of theligninase and cellulase fusion proteins occurs primarily in the leavesof the plant, and because the ligninase and cellulase fusion proteinseach has a signal peptide that directs its transport to plastids, theligninase and cellulase fusion proteins accumulate in the plastids.

In the preferred embodiment of the present invention, the 3′ ends of thenucleotide sequence encoding the above ligninase and cellulase fusionproteins are operably linked to a 3′ noncoding sequence wherein thenoncoding sequence contains a poly(A) cleavage/addition site and otherregulatory sequences which enables the RNA transcribed therefrom to beproperly processed and polyadenylated which in turn affects stability,transport and translation of the RNA transcribed therefrom in the plantcell. Examples of 3′ noncoding sequences include the 3′ noncodingsequence from the potato protease inhibitor II gene, which includesnucleotides 871 to 1241 of SEQ ID NO: 15 (GenBank Accession No. M151.86)and the 3′ noncoding sequence from the Agrobacterium nopaline synthasegene, which includes nucleotides 2001 to 2521 of SEQ ID NO:16 (GenBankAccession No. V00087 J01541).

The above heterologous gene expression cassettes can be constructedusing conventional molecular biology cloning methods. In a particularlyconvenient method, PCR is used to produce the nucleotide fragments forconstructing the gene expression cassettes. By using the appropriate PCRprimers, the precise nucleotide regions of the above DNAs can beamplified to produce nucleotide fragments for cloning. By furtherincluding in the PCR primers restriction enzyme cleavage sites which aremost convenient for assembling the heterogenous gene expressioncassettes (e.g., restriction enzyme sites that are not in the nucleotidefragments to be cloned), the amplified nucleotide fragments are flankedwith the convenient restriction enzyme cleavage sites for assembling thenucleotide fragments into heterogenous gene expression cassettes. Theamplified nucleotide fragments are assembled into the heterogeneous geneexpression cassettes using conventional molecular biology methods. Basedupon the nucleotide sequences provided herein, how to construct theheterogenous gene expression cassettes using conventional molecularbiology methods with or without PCR would be readily apparent to oneskilled in the art.

In a further embodiment of the present invention, the transgenic plantcomprises more than one heterogeneous gene expression cassette. Forexample, the transgenic plant comprises a first cassette which containsa DNA encoding a ligninase fusion protein, and one or more cassetteseach containing a DNA encoding a particular cellulase fusion protein.Preferably, both the ligninase and cellulase fusion proteins compriseamino acids of a signal peptide which directs the fusion proteins toplant organelles. In a preferred embodiment, the signal peptide for eachis the rbcS SP or the SKL motif.

In a further still embodiment, the transgenic plant comprises DNAencoding the ligninase fusion protein such as the ckg4 or ckg5 LIP, anendoglucanase fusion protein such as the e1 fusion protein, and acellobiohydrolase fusion protein such as the cbh1 fusion protein. In afurther still embodiment, the transgenic plant comprises DNA encodingthe ligninase fusion protein such as the ckg4 or ckg5 LIP, anendoglucanase fusion protein such as the e1 fusion protein, acellobiohydrolase fusion protein such as the cbh1 fusion protein, abeta-glucosidase, and a glucanhydrolase. Preferably, both the ligninaseand cellulase fusion proteins comprise amino acids of a signal peptidewhich directs the fusion proteins to plant organelles. In a preferredembodiment, the signal peptide for each is the rbcS SP or the SKL motif.

To make the transgenic plants of the present invention, plant materialsuch as meristem primordia tissue is transformed with plasmids, eachcontaining a particular heterogenous gene expression cassette using theBiolistic bombardment method as described in Example 5 and in U.S. Pat.No. 5,767,368 to Zhong et al. Further examples of the Biolisticbombardment method are disclosed in U.S. application Ser. No. 08/036,056and U.S. Pat. No. 5,736,369 to Bowen et al. Each heterogenous geneexpression cassette is separately introduced into a plant tissue and thetransformed tissue propagated to produce a transgenic plant thatcontains the particular heterogenous gene expression cassette. Thus, theresult is a transgenic plant containing the heterogenous gene expressioncassette expressing a ligninase such as ckg4 or ckg5, a transgenic plantcontaining a heterogenous gene expression cassette expressingendoglucanase such as e1, a transgenic plant containing a heterogenousgene expression cassette expressing a cellobiohydrolase such as cbh1, atransgenic plant containing a heterogenous gene expression cassetteexpressing an exoglucanase such as beta-glucosidase, and a transgenicplant containing a heterogenous gene expression cassette expressing anexoglucanase such as glucanhydrolase.

Alternatively, transformation of corn plants can be achieved usingelectroporation or bacterial mediated transformation using a bacteriumsuch as Agrobacterium tumefaciens to mediate the transformation of cornroot tissues (see Valvekens et al. Proc. Nat'l. Acad. Sci. USA. 85:5536-5540 (1988)) or meristem primordia.

In a preferred embodiment of the present invention, the transgenic plantcomprises one or more ligninase fusion proteins and one or morecellulase fusion proteins. Construction of the preferred transgenicplant comprises making first generation transgenic plants as above, eachcomprising a ligninase fusion protein, and transgenic plants as above,each comprising a cellulase fusion protein. After each first generationtransgenic plant has been constructed, progeny from each of the firstgeneration transgenic plants are cross-bred by sexual fertilization toproduce second generation transgenic plants comprising variouscombinations of both the ligninase fusion protein and the cellulasefusion protein.

For example, various combinations of progeny from the first generationtransgenic plants are cross-bred to produce second generation transgenicplants that contain ckg4 and cbh1, e1, beta-glucosidase, or ckg5; secondgeneration transgenic plants that contain ckg5 and cbh1, e1, orbeta-glucosidase; second generation transgenic plants that contain e1 orbeta glucosidase, and a second generation transgenic plant that containse1 and beta-glucosidase.

Progeny of the second generation transgenic plants are cross-bred bysexual fertilization among themselves or with first generationtransgenic plants to produce third generation transgenic plants thatcontain one or more ligninases, one or more cellulases, or combinationsthereof.

For example, cross-breeding a second generation transgenic plantcontaining ckg4 and cbh1 with a second generation transgenic plantcontaining e1 and beta-glucosidase produces a third generationtransgenic plant containing ckg4, cbh1, e1, and beta-glucosidase. Thethird generation transgenic plant can be cross-bred with a firstgeneration transgenic plant containing ckg5 to produce a fourthgeneration transgenic plant containing ckg4, ckg5, cbh1, e1, andbeta-glucosidase.

It will be readily apparent to one skilled in the art that othertransgenic plants with various combinations of ligninases and cellulasescan be made by cross-breeding progeny from particular transgenic plants.Zhang et al, Theor. Appl. Genet. 92: 752-761, (1996), Zhong et al, PlantPhysiol. 110: 1097-1107, (1996), and Zhong et al, Planta, 187: 483-489,(1992) provide methods for making transgenic plants by sexualfertilization.

Alternatively, plant material is transformed as above with a plasmidcontaining a heterologous gene expression cassette encoding theligninase fusion protein. The transgenic plant is recovered from theprogeny of the transformed plant material. Next, plant material from thetransgenic plant is transformed with a second plasmid containing aheterologous gene expression cassette encoding the cellulase fusionprotein and a second selectable marker. The transgenic plant isrecovered from the progeny of the transformed plant material. It will bereadily apparent to one skilled in the art that transgenic plantscontaining any combination of ligninases and cellulases can be made bythe above method.

In a preferred embodiment, the above heterologous gene expressioncassettes further include therein nucleotide sequences that encode oneor more selectable markers which enable selection and identification oftransgenic plants that express the modified cellulase of the presentinvention. Preferably, the selectable markers confers additionalbenefits to the transgenic plant such as herbicide resistance, insectresistance, and/or resistance to environmental stress.

Alternatively, the above transformations are performed byco-transforming the plant material with a first plasmid containing aheterologous gene expression cassette encoding a selectable marker and asecond plasmid containing a heterologous gene expression cassetteencoding a ligninase or cellulase fusion protein. The advantage of usinga separate plasmid is that after transformation, the selectable markercan be removed from the transgenic plant by segregation, which enablesthe selection method for recovering the transgenic plant to be used forrecovering transgenic plants in subsequent transformations with thefirst transgenic plant.

Examples of preferred markers that provide resistance to herbicidesinclude, but are not limited to, the bar gene from Streptomyceshygroscopicus encoding phosphinothricin acetylase (PAT), which confersresistance to the herbicide glufonsinate; mutant genes which encoderesistance to imidazalinone or sulfonylurea such as genes encodingmutant form of the ALS and AHAS enzyme as described by Lee at al. EMBOJ. 7: 1241 (1988) and Miki et al., Theor. Appl. Genet. 80: 449 (1990),respectively, and in U.S. Pat. No. 5,773,702 to Penner et al.; geneswhich confer resistance to glycophosphate such as mutant forms of EPSPsynthase and aroA; resistance to L-phosphinothricin such as theglutamine synthetase genes; resistance to glufosinate such as thephosphinothricin acetyl transferase (PAT and bar) gene; and resistanceto phenoxy proprionic acids and cycloshexones such as the ACCAseinhibitor-encoding genes (Marshall et al. Theor. Appl. Genet. 83: 435(1992)). The above list of genes which can import resistance to anherbicide is not inclusive and other genes not enumerated herein butwhich have the same effect as those above are within the scope of thepresent invention.

Examples of preferred genes which confer resistance to pests or diseaseinclude, but are not limited to, genes encoding a Bacillus thuringiensisprotein such as the delta-endotoxin, which is disclosed in U.S. Pat. No.6,100,456 to Sticklen et al.; genes encoding lectins, (Van Damme et al.,Plant Mol. Biol. 24: 825 (1994)); genes encoding vitamin-bindingproteins such as avidin and avidin homologs which can be used aslarvicides against insect pests; genes encoding protease or amylaseinhibitors, such as the rice cysteine proteinase inhibitor (Abe et al.,J. Biol. Chem. 262: 16793 (1987)) and the tobacco proteinase inhibitor I(Hubb et al., Plant Mol. Biol. 21: 985 (1993)); genes encodinginsect-specific hormones or pheromones such as ecdysteroid and juvenilehormone, and variants thereof, mimetics based thereon, or an antagonistsor agonists thereof; genes encoding insect-specific peptides orneuropeptides which, upon expression, disrupts the physiology of thepest; genes encoding insect-specific venom such as that produced by awasp, snake, etc.; genes encoding enzymes responsible for theaccumulation of monoterpenes, sesquiterpenes, asteroid, hydroxamincacid, phenylpropanoid derivative or other non-protein molecule withinsecticidal activity; genes encoding enzymes involved in themodification of a biologically active molecule (see U.S. Pat. No.5,539,095 to Sticklen et al., which discloses a chitinase that functionsas an anti-fungal); genes encoding peptides which stimulate signaltransduction; genes encoding hydrophobic moment peptides such asderivatives of Tachyplesin which inhibit fungal pathogens; genesencoding a membrane permease, a channel former or channel blocker (forexample cecropin-beta lytic peptide analog renders transgenic tobaccoresistant to Pseudomonas solanacerum) (Jaynes et al. Plant Sci. 89: 43(1993)); genes encoding a viral invasive protein or complex toxinderived therefrom (viral accumulation of viral coat proteins intransformed cells of some transgenic plants impart resistance toinfection by the virus the coat protein was derived as shown by Beachyet al. Ann. Rev. Phytopathol. 28: 451 (1990); genes encoding aninsect-specific antibody or antitoxin or a virus-specific antibody(Tavladoraki et al. Nature 366: 469 (1993)); and genes encoding adevelopmental-arrestive protein produced by a plant, pathogen orparasite which prevents disease. The above list of genes which canimport resistance to disease or pests is not inclusive and other genesnot enumerated herein but which have the same effect as those above arewithin the scope of the present invention.

Examples of genes which confer resistance to environmental stressinclude, but are not limited to, mtld and HVA1, which are genes thatconfer resistance to environmental stress factors; rd29A and rd19B,which are genes of Arabidopsis thaliana that encode hydrophilic proteinswhich are induced in response to dehydration, low temperature, saltstress, or exposure to abscisic acid and enable the plant to toleratethe stress (Yamaguchi-Shinozaki et al., Plant Cell 6: 251-264 (1994)).Other genes contemplated can be found in U.S. Pat. Nos. 5,296,462 and5,356,816 to Thomashow. The above list of genes, which can importresistance to environmental stress, is not inclusive and other genes notenumerated herein but which have the same effect as those above arewithin the scope of the present invention.

Thus, it is within the scope of the present invention to providetransgenic plants which express one or more ligninase fusion proteins,one or more cellulase fusion proteins, and one or more of anycombination of genes which confer resistance to an herbicide, pest, orenvironmental stress.

In particular embodiments of the present invention, the heterologousgene expression cassettes can further be flanked with DNA containing thematrix attachment region (MAR) sequence. While use of MAR in the presentinvention is optional, it can used to increase the expression level oftransgenes, to get more reproducible results, and to lower the averagecopy number of the transgene (Allen et al., The Plant Cell 5: 603-613(1993); Allen et al., The Plant Cell 8: 899-913 (1996); Mlynarova etal., The Plant Cell 8: 1589-1599 (1996)).

To degrade the lignocellulose in the leaves and stalks of the transgenicplants of the present invention, the transgenic plant is ground up toproduce a plant material using methods currently available in the art todisrupt a sufficient number of the plant organelles containing theligninase and cellulase therein. The ligninase and cellulase degrade thelignocellulose of the transgenic plant into fermentable sugars,primarily glucose, and residual solids. The fermentable sugars are usedto produce ethanol or other products.

The transgenic plants can be processed to ethanol in an improvement onthe separate saccharification and fermentation (SHF) method (Wilke etal., Biotechnol. Bioengin. 6: 155-175 (1976)) or the simultaneoussaccharification and fermentation (SSF) method disclosed in U.S. Pat.No. 3,990,944 to Gauss et al. and U.S. Pat. No. 3,990,945 to Huff et al.The SHF and SSF methods require pre-treatment of the plant materialfeedstock with dilute acid to make the cellulose more accessiblefollowed by enzymatic hydrolysis using exogenous cellulases to produceglucose from the cellulose, which is then fermented by yeast to ethanol.In some variations of the SHF or SSF methods, the plant material ispre-treated with heat or with both heat and dilute acid to make thecellulose more accessible.

An SHF or SSF method that uses the transgenic plant material of thepresent invention as the feedstock is an improvement over the SHF or SSFmethod because the transgenic plant material contains its own cellulasesand ligninases or cellulases. Therefore, exogenous ligninases and/orcellulases do not need to be added to the feedstock. Furthermore,because particular embodiments of the transgenic plant material produceligninase, the need for pre-treatment of the plant material in thoseembodiments before enzymatic degradation is not necessary. In a furtherimprovement over the SHF method, the transgenic plant material is mixedwith non-transgenic plant material and the mixture processed to ethanol.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding acellulase fusion protein and a heterologous gene expression cassettecomprising a DNA encoding the bar gene (Table 1).

TABLE 1 Construct Plasmid features 1 rbcSP/e1/pin 3′//Act1 rbcSPleaf-specific promoter driving P/bar/nos 3′ cellulase cDNA of A.cellulolyticus 2 rbcSP/cbh1/pin 3′//Act1 rbcSP leaf-specific promoterdriving P/bar/nos 3′ cellulase cDNA of T. reesi 3 rbcSP/rbcS SP/e1/pin3′//Act1 The rbcS SP targets cellulase P/bar/nos 3′ of A. cellulolyticusinto maize chloroplasts 4 rbcSP/rbcS SP/cbh1/pin The rbcS SP targetscellulase of 3′//Act1 P/bar/nos 3′ T. reesi into maize chloroplastsAbbreviations:

The term “rbcSP” means the rice rubisco rbcS promoter region. The rbcSPis a leaf-specific promoter that limits transcription of rbcS to theleaves (Schaeffer and Sheen, Plant Cell 3: 997-1012 (1991)). Thenucleotide sequence for the rbcS promoter region is set forth in SEQ IDNO:1.

The term “e1” means the cDNA isolated from Acidothermus cellulolyticuswhich encodes the cellulase EI beta-1,4-endoglucanase precursor. Thenucleotide sequence for the gene encoding e1 is set forth in SEQ IDNO:4. In this example, the codons for the 41 amino acid leader sequence(nucleotides 824 to 946 of SEQ ID NO:4) are removed.

The term “cbh1” means the cDNA isolated from Trichoderma reesi thatencodes the cellulase cellobiohydrolase. The nucleotide sequence for thegene encoding cbh1 is set forth in SEQ ID NO:10. In this example, thecodons for the 54 amino acid leader sequence (nucleotides 210 to 671 ofSEQ ID NO:10) are removed.

The term “pin3′” means the potato protease inhibitor II-chloramphenicolacetyltransferase gene's 3′ untranslated sequence which containstranscription termination signals (Thornburg et al., Proc. Natl. Acad.Sci. USA 84: 744-748 (1987)). The pin3′ untranslated sequence includesnucleotides 882 to 1241 of the nucleotide sequence set forth in SEQ IDNO: 15.

The term “bar” means the phosphinothricin acetyl transferase gene(Thompson et al., EMBO J. 6: 2519-2523 (1987)). The bar gene is aselectable marker for herbicide resistance. The 5′ end of bar isoperably linked to the rice actin 1 gene promoter which has been shownto operable in maize (Zhong et al., Plant Physiology 110: 1097-1107(1996); Zhang et al., Theor. Appl. Genet. 92: 752-761 (1996); Zhang etal., Plant Science 116: 73-84 (1996)). The 3′ end of bar is operablylinked to the nos 3′ untranslated sequences. The nucleotide sequence ofthe bar gene is set forth in SEQ ID NO:18 (GenBank Accession No.X05822), which encodes the bar having the amino acid sequence fromnucleotides 160 to 711.

The term “Act1 P” means the rice Act1 gene promoter which furtherincludes the 5′ intron region (McElroy et al., Mol. Gen. Genet. 231:150-160 (1991). The sequence of the Act1 gene and its promoter is setforth in SEQ ID NO:19 (GenBank Accession No. X63830).

The term “nos3′” means the 3′ untranslated sequence from theAgrobacterium nopaline synthase gene encoding nopaline synthase of theamino acid sequence as set forth in SEQ ID NO:17 which includesnucleotides 2002 to 2521 of SEQ ID NO:16 (GenBank Accession No. VOOO87J01541). The Nos3′ sequence contains transcription termination signals.

The term “rbcS SP” means the rice rubisco small subunit signal peptidewhich consists of 47 codons encoding the peptide with the amino acidsequence set forth in SEQ ID NO:2. The rbcS SP directs the translocationof the rbcS small subunit or any polypeptide to which it is attached tothe chloroplasts (Loza-Tavera et al., Plant Physiol. 93: 541-548(1990)).

Construct 1 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the cellulase encoded by e1 to the cells of theleaves of the maize plant.

Construct 2 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the cellulase encoded by cbh1 to the cells of theleaves of the maize plant.

Construct 3, which is shown in FIG. 1, is like construct 1 except thatDNA encoding the rbcS SP signal peptide is operably linked to the 5′ endof the e1, and construct 4, which is shown in FIG. 2, is like construct2 except that DNA encoding the rbcS SP signal peptide is operably linkedto the 5′ end of cbh1. Therefore, expression of cellulase from construct3 or 4, which is limited to the cells of the leaves, directed to thechloroplasts in the cells. All of the above constructs are adjacent to aheterologous gene expression cassette containing the bar gene operablylinked to the Act1 promoter.

Construction of plasmid rbcSP/rbcS SP/cbh1//pin3′//Act1 P/bar/nos3′. Thestarting plasmid was pBR10-11 which contained the crylA(b) gene upstreamof the pin3′. Between the crylA(b) and the pin3′ is a DNA polylinkercontaining in the following order a SmaI, BamHI, SpeI, XbaI, NotI, andEagI restriction enzyme recognition site. The plasmid pBR10-11(available from Silan Dai and Ray Wu, Department of Molecular Biologyand Genetics, Biotechnology Building, Cornell University, Ithaca, N.Y.14853-2703) was digested with restriction enzymes SpeI and XbaI toproduce a 9.2 kb DNA fragment. The 9.2 kb DNA fragment(pBR10-11/SpeI/XbaI/9.2 kb fragment) was purified by agarose gelelectrophoresis.

The plasmid pB210-5a (available from William S. Adney, Mike Himmel, andSteve Thomas, National Renewable Energy Laboratory, 1670 Cole Boulevard,Golden Colo. 80401) containing the cbh1 gene from Trichoderma reesei(Trichoderma longibrachiatum) was digested with SpeI and XbaI. Thedigested plasmid was electrophoresed on an agarose gel and a 1.8 kbfragment (pB210-5a/SpeI/XbaI/1.8 kb fragment containing cbh1) waspurified from the gel.

The above 9.2 kb and the 1.8 kb DNA fragments were ligated togetherusing T4 DNA ligase to make plasmid “pBR10-11-cbh1” which was used totransform E. coli XL1 Blue. Transformed bacteria containing plasmidpBR10-11-cbh1 were identified by plating on LB agar gels containingampicillin.

The plasmid pBR10-11-cbh1 was digested with SmaI and PstI. The PstI endwas made blunt with mung bean exonuclease. The digested plasmid waselectrophoresed on an agarose gel and the 2.8 kb DNA fragment containingcbh1 and pin3′ was purified from the gel. The purified DNA fragment wasdesignated “cbh1-pin3′/blunt-ended.”

The plasmid pDM302 (Cao et al., Plant Cell Reports 11: 586-591 (1992)),shown in FIG. 3, containing upstream of a ClaI site, a gene cassetteconsisting of the bar gene flanked by an upstream Act1 promoter and adownstream nos3′, was digested with ClaI. The ClaI ends of the digestedplasmid were made blunt with Taq DNA polymerase and the digested plasmidelectrophoresed on an agarose gel. The digested plasmid was designated“pDM302/ClaI/blunt-ended.”

The pDM302/ClaI/blunt-ended plasmid and the cbh1-pin3′/blunt-ended DNAfragment were ligated together using T4 DNA ligase to make plasmid“pDM302-cbh1-pin3′” which was used to transform E. coli XL1Blue.Transformed bacteria containing plasmid pDM302-cbh1-pin3′ wereidentified by plating on LB agar gels containing ampicillin.

Plasmid pDM302-cbh1-pin3′ was digested with SpeI, the ends made bluntwith Taq DNA polymerase, and purified by agarose gel electrophoresis.The purified DNA fragment was designated“pDM302-cbh1-pin3′/SpeI/blunt-ended.”

Plasmid pRRI (available from Silan Dai and Ray Wu, Department ofMolecular Biology and Genetics, Biotechnology Building, CornellUniversity, Ithaca, N.Y. 14853-2703), which contains the rice rbcS smallsubunit gene, was digested with PstI. The rbcS promoter is flanked byPstI sites. The PstI ends were made blunt with mung bean nuclease andthe 2 kb DNA fragment (rice rbcS/PstI/blunt-ended) containing thepromoter was purified by agarose gel electrophoresis.

Rice rbcSP/PstI/blunt-ended and plasmid pDM-cbh1-pin3′/SpeI/blunt-endedwere ligated using T4 DNA ligase to makerbcSP/cbh1/pin3′//Act1P/bar/nos3′ which was then used to transform E.coli XL Blue. Transformed bacteria containing plasmidrbcSP/cbh1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

PCR was used to insert NotI sites intorbcSP/cbh1/pin3′//Act1P/bar/nos3′. These sites were used to insert therice rubisco signal peptide in place of the cbh1 signal peptide. ThepRRI plasmid was the source of the rice rubisco signal peptide. It wasalso the used as a PCR template to produce the PCR product containingthe rice rubisco signal peptide flanked by NotI cohesive termini. Therice rubisco signal peptide and the rbcSP/cbh1/pin3′//Act1P/bar/nos3′plasmid were ligated together using T4 DNA ligase to make rbcSP/rbcSSP/cbh1/pin3′//Act1P/bar/nos3′ which was then used to transform E. coliXL Blue. Transformed bacteria containing plasmid rbcSP/rbcSSP/cbh1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

Construction of plasmid rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′. PlasmidpMPT4-5 (available from William S. Adney, Mike Himmel, and Steve Thomas,national Renewable Energy laboratory, 1670 Colorado Boulevard, Golden,Colo. 80401) contains the e1 gene encoding endoglucanase I fromAcidothermus cellulolyticus as a 3.7 kb PvuI DNA fragment cloned intopGEM7 (Promega Corporation, Madison, Wis.). PCR was used to produce aDNA fragment containing the e1 gene flanked by AscI recognition sites.Plasmid rbcSP/cbh1/pin3′//Act1P/bar/nos3′ was also mutagenized by PCR tointroduce AscI sites flanking the cbh1 gene. Next, the plasmidrbcSP/cbh1/pin3′//Act1P/bar/nos3′ was digested with AscI and the plasmidfree of the cbh1 gene was purified by agarose gel electrophoresis. TheAscI flanked e1 gene was ligated using T4 DNA ligase into therbcSP/cbh1/pin3′//Act1P/bar/nos3′ free of the cbh1 gene to produceplasmid rbcSP/e1/pin3′//Act1P/bar/nos3′, which then used to transform E.coli XL Blue. Transformed bacteria containing plasmidrbcSP/e1/pin3′//Act1P/bar/nos3′ were identified by plating on LB agargels containing ampicillin.

PCR was used to insert NotI sites into rbcSP/e1/pin3′//Act1P/bar/nos3′.These sites were used to insert the rice rubisco signal peptide in placeof the cbh1 signal peptide. The pRRI plasmid was the source of the ricerubisco signal peptide. It was also the used as a PCR template toproduce the PCR product containing the rice rubisco signal peptideflanked by NotI cohesive termini. The rice rubisco signal peptide andthe rbcSP/e1/pin3′//Act1P/bar/nos3′ plasmid were ligated together usingT4 DNA ligase to make rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′ which wasthen used to transform E. coli XL Blue. Transformed bacteria containingplasmid rbcSP/rbcS SP/e1/pin3′//Act1P/bar/nos3′ were identified byplating on LB agar gels containing ampicillin.

Both heterologous gene expression cassettes are contiguous and thecontiguous cassettes can be flanked by MAR sequences.

EXAMPLE 2

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding acellulase fusion protein. The plasmid constructs are shown in Table 2.

TABLE 2 Construct Plasmid features 1 rbcSP/cbh1/pin 3′ rbcSPleaf-specific promoter driving cellulase cDNA of T. reesei 2 rbcSP/rbcSSP/cbh1/pin 3′ The rbcS SP targets cellulase of T. reesi into maizechloroplasts 3 rbcSP/rbcS SP/syn- The rbcS SP targets modified cellulasecbh1/pin 3′ of T. reesei into maize chloroplasts 4 CaMv35s/SSU/e1/nos3′The SSU targets the cellulase of A. cellulolyticus into maizechloroplasts 5 CaMv35s/VSP/e1/nos3′ The VSP targets the cellulase of A.cellulolyticus into maize apoplasts 6 CaMv35s/e1/nos3′ No signal peptideAbbreviations:

The term “syn-cbh1” refers to a cbh1 gene that has been codon-modifiedfor use in transformation of tobacco plants. It is available from.

The term “CaMV35s” refers to the cauliflower mosaic virus promoter.

The term “SSU” refers to the glycine max rbcS signal peptide. Glycinemax is a soybean and not a rice variety.

The term “VSP” refers to the soybean vegetative storage protein betasignal peptide.

The remainder of the terms in Table 2 are the same as those for table 1.

Construct 1, which is shown in FIG. 4, is plasmid pSMF13 which isplasmid pSK (Stratagene, La Jolla, Calif.) which contains cbh1 operablylinked to the rice rubisco rbcS leaf-specific promoter which limitsexpression of the cellulase encoded by cbh1 to the cells of the leavesof the maize plant.

Construct 2, which is shown in FIG. 5, is plasmid pSF15 which is plasmidpSK which contains cbh1 operably linked to the rice rubisco rbcSleaf-specific promoter which limits expression of the cellulase encodedby cbh1 to the cells of the leaves of the maize plant and a DNA encodingthe rbcS SP which targets the cellulase to the chloroplasts.

Construct 3, which is shown in FIG. 6, is like construct 2 except thatthe cbh1 has been modified to decrease the GC content of the cbh1 to anamount similar to the GC content of the tobacco plant genome. Thenucleotide sequence of the modified cbh1 (syn-cbh1) in plasmid pBI221 isset forth in SEQ ID NO:20.

Construct 4, which is shown in FIG. 7, is plasmid pTZA8 which is plasmidpBI121 which contains the caMV35s promoter, which is a constitutivepromoter that is active in most plant tissues, to drive expression of e1which is operably linked to a DNA encoding the SSU signal peptide whichtargets the cellulase to the chloroplasts.

Construct 5, which is shown in FIG. 8, is plasmid pZA9 which is similarto construct 4 except the signal peptide is encoded by DNA encoding theVSP signal peptide which targets the cellulase to the apoplasts.Construct 6, which is shown in FIG. 9, is plasmid pZA10 which is similarto construct 4 or 5 except that e1 is not operably linked to a DNAencoding a signal peptide that targets the cellulase to a plantorganelle.

The constructs were prepared as follows.

First, the plasmid pRR1, which contains the rice rbcS gene was obtainedfrom Ray Wu and Silan Dai, Cornell University. The rice rubisco (rbcS)small subunit was cleaved from pRR1 using EcoRI and EcoRV restrictionsites to release a 2.1 kb DNA fragment containing the rbcS. The 2.1 kbDNA fragment was ligated into the plasmid pSK between the EcoRI andEcoRV sites to produce plasmid pSMF8. The 2.1 kb DNA fragment providedthe promoter for the cbh1 constructs below.

Next, the cbh1 gene was cloned downstream of rbcS promoter in plasmidpSMF8. First, a 1.7 kb DNA fragment containing the cbh1 gene fromTrichoderma reesei was isolated from plasmid pB210-5A (available fromWilliam S. Adney, Mike Himmel, and Steve Thomas, National RenewableEnergy Laboratory; described in Shoemaker et al., Bio/Technology 1:691-696 (1983)) by digesting with SalI and XhoI. The ends of the 1.7 kbDNA fragment were made blunt end using DNA polymerase I (largefragment). The blunt-ended DNA fragment was cloned into plasmid pSMF8,which had been digested with BamHI and the ends made blunt with DNApolymerase I, to make plasmid pSMF9.

Next, to complete the heterologous gene expression cassette, the pin3′transcription termination nucleotide sequence was inserted at the 3′ endof the cbh1 gene in plasmid pSMF9. The pin3′ transcription terminationnucleotide sequence was cleaved from pBR10-11 with PstI. However, toremove the pin3′ transcription termination nucleotide sequence frompBR10-11, a PstI site had to be introduced upstream of the pin3′transcription termination nucleotide sequence.

To generate the PstI site upstream of the pin3′ transcriptiontermination nucleotide sequence in pBR10-11, the pBR10-11 was digestedwith NotI and XhoI and a 70 bp multi-cloning site nucleotide sequence,which had been isolated from the pSK vector by digesting with NotI andXhoI, was cloned between the NotI and XhoI sites of the pBR10-11 toproduce plasmid pSMF11. The pin3′ transcription termination nucleotidesequence was then removed from pSMF11 by digesting with PstI to producea 1 kb DNA fragment which was then cloned into the PstI site of pSK,which had been digested with PstI, to produce plasmid pSMF12. PSMF12 wasthen digested with NotI to produce a 1 kb DNA fragment containing thepin3′ transcription termination nucleotide sequence. The 1 kb DNAfragment cloned into the NotI site downstream of the cbh1 gene in pSMF9,which had been digested with NotI, to produce plasmid pSMF13 (construct1 in Table 2).

Next, a DNA encoding a signal peptide which targets proteins to which itis attached to the chloroplasts was inserted upstream of the cbh1 and inthe same reading frame as the cbh1. Thus, a fusion protein is producedfrom translation of RNA transcribed from the cbh1 DNA linked to the DNAencoding the signal peptide. DNA encoding the signal peptide (SP) wasisolated from the rbcS in the pRRI plasmid. Because there were noconvenient restriction enzyme sites available which flanked the DNAencoding the SP for cloning, it was planned to PCR amplify that regioncontaining the DNA encoding the SP using PCR primers with PCR primersthat contained convenient restriction enzyme sites for cloning. At theend of the rbcS promoter pSMF13 is a unique AvrII site and upstream ofthe first ATG of the cbh1 gene is a unique BsrGI. A DNA encoding the SPthat was flanked with an AvrII site on one end and a BsrGI site on theopposite end would be able to be cloned between the AvrII and BsrGIsites in PSMF13. That would place the DNA encoding the SP between therbcS promoter and the cbh1 gene and would enable a fusion proteincontaining the SP fused to the cellulase.

Therefore, PCR primers were synthesized using DNA sequences for theAvrII and BsrGI sites and the SP DNA sequences. The upstream PCR primer(SP1F) had the nucleotide sequence 5′-CCGCCTAGGCGCATGGCCCCCTCCGT-3′ (SEQID NO:21) and the downstream PCR primer (SP3R) had the nucleotidesequence 5′-CGCTGTACACGCACCTGATCCTGCC-3′ (SEQ ID NO:22). Plasmid pRR1encoding the SP was PCR amplified with the PCR primers and the 145 bpamplified product was purified using 2% agarose gel. The purified 145 bpproduct was sequenced to confirm that the 145 bp amplified productcontained the SP nucleotide sequences. The amplified product wasdigested with AvrII and BsrGI and cloned between the AvrII and BsrGIsites of pSMF13 digested with AvrII and BsrGI to produce plasmid pSMF14.

To produce pSMF15 which contains a cbh1 gene codon-modified to decreasethe GC content of the cbh1 gene to an amount similar to the GC contentof the tobacco genome, a synthetic cbh1 (syn-cbh1) gene was obtainedfrom plasmid pZD408 (available from Ziyu Dai, Pacific Northwest nationalLaboratory, 902 Battelle Boulvard, Richland, Wash. 99352). The syn-cbh1is a cbh1 which had been codon-modified for use in tobacco planttransformations. The nucleotide sequence of syn-cbh1 is set forth in SEQID NO:20. Plasmid pZD408 was linearized with NcoI and the ends madeblunt. Then, the blunt-ended pZD408 was digested with HindIII to removethe CaMV35S promoter. A 4.5 kb DNA fragment containing the syn-cbh1 wasisolated from the CaMV35S promoter by agarose gel electrophoresis. The4.5 kb DNA fragment was dephosphorylated and the DNA fragment containinga blunt end and a HindIII end was named pZD408B.

Plasmid pSMF14 was digested with BsrGI, the BsrGI ends made blunt, andthen pSMF14 was digested with HindIII to produce a DNA fragmentcontaining the rbcS promoter with the DNA encoding the SP flanked by ablunt end and a HindIII end. The DNA fragment was purified by agarosegel electrophoresis and ligated to the pZD408B DNA fragment to produceplasmid pSMF15 (construct 3 of Table 2).

The heterologous gene expression cassettes are contiguous can be flankedby MAR sequences.

EXAMPLE 3

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding aligninase fusion protein and a heterologous gene expression cassettecomprising a DNA encoding the bar gene. The constructs are shown inTable 3.

TABLE 3 Construct Plasmid features 1 rbcSP/ckg4/pin 3′//Act1 rbcSPleaf-specific promoter driving P/bar/nos 3′ ckg4 cDNA of P.chrysosporium 2 rbcSP/ckg5/pin 3′//Act1 rbcSP leaf-specific promoterdriving P/bar/nos 3′ ckg5 cDNA of P. chrysosporium 3 rbcSP/rbcSSP/ckg4/pin The rbcS SP targets ckg4 into maize 3′//Act1 P/bar/nos 3′chloroplasts 4 rbcSP/rbcS SP/ckg5/pin The rbcS SP targets ckg5 intomaize 3′// Act1 P/bar/nos 3′ chloroplastsAbbreviations:

The terms “ckg4” and “ckg5” mean the ligninase cDNAs isolated from thebasidiomycete Phanerochaete. chrysosporium, SEQ ID NO:11 and SEQ IDNO:13, respectively. The codons for the 28 amino acid leader are deletedso that the expressed gene product remains inside the cells.

The remainder of the terms in Table 3 are the same as those for Table 1.All plasmid constructs contain the selectable marker gene (bar) drivenby the rice actin 1 gene promoter. The rice actin gene and its promoterare disclosed in U.S. Pat. No. 5,641,876 to McElroy et al.

Construct 1 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the ligninase encoded by ckg4 to the cells of theleaves of the maize plant.

Construct 2 contains the rice rubisco rbcS leaf-specific promoter whichlimits expression of the ligninase encoded by ckg5 to the cells of theleaves of the maize plant.

Construct 3, which is shown in FIG. 10, contains the rice rubisco rbcSleaf-specific promoter which limits expression of the ligninase encodedby ckg4 to the cells of the leaves of the maize plant and furthercontains DNA encoding the rbcS SP which targets the ligninase to thechloroplasts.

Construct 4, which is shown in FIG. 11, contains the rice rubisco rbcSleaf-specific promoter which limits expression of the ligninase encodedby ckg5 to the cells of the leaves of the maize plant and furthercontains DNA encoding the rbcS SP which targets the ligninase to thechloroplasts. All of the above constructs are adjacent to a heterologousgene expression cassette containing the bar gene operably linked to theAct1 promoter. Both heterologous gene expression cassettes arecontiguous and the contiguous cassettes can be flanked by MAR sequences.

EXAMPLE 4

This example shows the construction of plasmids comprising aheterologous gene expression cassette comprising a DNA encoding aligninase fusion protein. The constructs are shown in Table 4.

TABLE 4 Construct Plasmid features 1 rbcSP/ckg4/pin 3′ rbcSPleaf-specific promoter driving ckg4 cDNA of P. chrysosporium 2rbcSP/ckg5/pin 3′ rbcSP leaf-specific promoter driving ckg5 cDNA of P.chrysosporium 3 rbcSP/rbcS SP/ckg4/pin 3′ The rbcS SP targets ckg4 intomaize chloroplasts 4 rbcSP/rbcS SP/ckg5/pin 3′ The rbcS SP targets ckg5into maize chloroplasts

The terms in table 4 are the same as those for Tables 1 and 3.

Construct 1, which is shown in FIG. 12, is plasmid pSMF18 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg4 to the cells ofthe leaves of the maize plant.

Construct 2, which is shown in FIG. 13, is plasmid pSMF19 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg5 to the cells ofthe leaves of the maize plant.

Construct 3, which is shown in FIG. 14, is plasmid pMSF16 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg4 to the cells ofthe leaves of the maize plant and further contains DNA encoding the rbcSSP which targets the ligninase to the chloroplasts.

Construct 4, which is shown in FIG. 15, is plasmid pSMF17 which isplasmid pSK which contains the rice rubisco rbcS leaf-specific promoterwhich limits expression of the ligninase encoded by ckg5 to the cells ofthe leaves of the maize plant and further contains DNA encoding the rbcSSP which targets the ligninase to the chloroplasts. The aboveheterologous gene expression cassettes can be flanked by MAR sequences.

The ligninase constructs shown in Table 4 are prepared as describedbelow.

Two plasmids, pCLG4 and pCLG5, the former containing a cDNA cloneencoding the ligninase gene ckg4 and the latter containing a cDNA cloneencoding the ckg5 were obtained from Dr. C. Adinarayana Reddy,Department of Microbiology and Public Health, Michigan State Universityand described in de Boer et al., Gene 6: 93-102 (1987), Corrigendum inGene 69: 369 (1988). These ligninase cDNA clones were prepared from awhite-rot filamentous fungus (Phanerochaete chrysosporium). The cDNAsfor ckg4 and ckg5 had each been cloned into the PstI site of the pUC9plasmid to make pCLG4 and pCLG5, respectively. The codons for the28-amino acid leader sequence is deleted from both cDNAs before cloningso that expressed gene product remains inside the cell.

Plasmid pSMF16 is made as follows. The ckg4 gene is removed from pCLG4by digesting the plasmid with the restriction enzymes XbaI and BstEII toproduce a 1.2 kb DNA fragment containing the ckg4 without the nucleotidesequence encoding the transit peptide. The BstEII removes the nucleotidesequences encoding the transit peptide of the ligninase.

The ends of the DNA fragment containing the ckg4 gene are made blunt andthe blunt-ended DNA fragment is ligated into pSMF14 in which the cbh1has been removed by digesting with BsrGI and XhoI and the ends madeblunt to produce pSMF16.

Plasmid pSMF18 is made as follows. The nucleotide sequence encoding therbcS signal peptide and cbh1 are removed from pSMF14 by digesting pSMF14with AvrII and XhoI instead of BsrGI and XhoI. The ends of the digestedpSMF14 are made blunt and the blunt-ended DNA fragment containing theckg4 gene, prepared as above, is ligated into the digested pSMF14 tomake plasmid pSMF18.

Plasmid pSMF17 is made as follows. The ckg5 gene is removed from pCLG5by digesting the plasmid with the restriction enzymes XbaI and EagI toproduce a 1.2 kb DNA fragment containing the ckg5 without the nucleotidesequence encoding the transit peptide. The EagI removes the nucleotidesequences encoding the transit peptide of the ligninase.

The ends of the DNA fragment containing the ckg5 are made blunt and theblunt-ended DNA fragment is ligated into pSMF14 in which the cbh1 hasbeen removed by digesting with BsrGI and XhoI and the ends made blunt toproduce pSMF17.

Plasmid pSMF19 is made as follows. The nucleotide sequence encoding therbcS signal peptide and cbh1 are removed from pSMF14 by digesting pSMF14with AvrII and XhoI instead of BsrGI and XhoI. The ends of the digestedpSMF14 are made blunt and the blunt-ended DNA fragment containing theckg5 gene, prepared as above, is ligated into the digested pSMF14 tomake plasmid pSMF19.

EXAMPLE 5

This example shows the transformation of maize multi-meristem primordiavia Biolistic bombardment with the plasmid constructs of Examples 1-4,regeneration of the transgenic plants, confirmation of the integrationof the plasmid constructs into the plant genome, and confirmation of theexpression of the cellulase or ligninase fusion proteins in thetransgenic plant. For transformations with the constructs of Examples 2and 4, which do not contain a selectable marker, a selectable markercomprising the bar gene in the plasmid pDM302 (Cao et al., Plant CellReports 11: 586-591 (1992)) is cotransfected into the cells with theplasmid containing the ligninase or cellulase heterologous geneexpression cassette.

Maize seeds have been germinated in Murashige and Skoog (MS) medium(Murashige and Skoog, Physiol. Plant 15: 473-497 (1962)) supplementedwith the appropriate growth regulators (Zhong et al., Planta 187:490-497 (1992)). Shoot meristems have been dissected and cultured for2-3 weeks until an initial multiplication of meristem have been producedfor bombardment.

The multi-meristem primordia explants are bombarded with tungstenparticles coated with particular plasmids of Example 1 or 3 or withparticular plasmids of Example 2 or 4 along with the plasmid containingthe heterogenous gene expression cassette containing the bar gene. Thebombarded explants are gently transferred onto meristem multiplicationmedium for further multiplication, about 6 to 8 more weeks. This step isrequired to reduce the degree of chimerism in transformed shoots priorto their chemical selection. Shoots are transferred to the above mediumcontaining 5 to 10 mg per liter glufosinate ammonium (PPT) selectablechemical for another 6 to 8 weeks. Chemically selected shoots are rootedin rooting medium containing the same concentration of PPT. Plantletsare transferred to pots, acclimated, and then transferred to agreenhouse.

When the plantlets or shoots are small, the quantity of transgenic plantmaterial is insufficient for providing enough DNA for Southern blothybridization; therefore, polymerase chain reaction (PCR) is used toconfirm the presence of the plasmid constructs the plantlets. Theamplified DNA produced by PCR is resolved by agarose or acrylamide gelelectrophoresis, transferred to membranes according standard Southerntransfer methods, and probed with the appropriate DNA construct orportion thereof according to standard Southern hybridization methods.Those shoots or plantlets which show they contain the construct in itsproper form are considered to have been transformed. The transformedshoots or plantlets are grown in the greenhouse to produce sufficientplant material to confirm that the plasmid constructs has been properlyintegrated into the plant genome. To confirm proper integration of theplasmid constructs into the plant genome, genomic DNA is isolated fromthe greenhouse grown transgenic plants and untransformed controls andanalyzed by standard Southern blotting methods as in Zhong et al., PlantPhysiology 110: 1097-1107 (1996); Zhang et al., Theor. Appl. Genet. 92:752-761 (1996); Zhang et al., Plant Science 116: 73-84 (1996); and,Jenes et al., In Transgenic Plants. Vol. 1. Kung, S-D and Wu, R (eds.).Academic Press, San Diego, Calif. pp. 125-146 (1992).

To confirm expression of the ligninase or cellulase fusion protein,total cellular RNA is isolated from the greenhouse grown plant tissuesas described in Zhong et al., Plant Physiology 110: 1097-1107 (1996).The mRNA encoding the cellulase or ligninase fusion protein is detectedby RNA Northern blot analysis using the same probes used for theSouthern blot analyses. Briefly, the RNA is electrophoresed on adenaturing formaldehyde agarose gel, transferred to nitrocellulose ornylon membranes, hybridized to the appropriate ligninase or cellulaseprobe, and then exposed to X-ray autoradiology film. The hybridizationbands are scanned using a densitometer which enables determination ofthe expression level of the specific mRNA.

Translation of the mRNA is confirmed by Western blot analysis accordingto the standard methods of Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350 (1979) and Burnette, Anal. Biochem. 112: 195 (1981) usingantibodies specific for ligninase or cellulase.

EXAMPLE 6

Transgenic maize containing both a ligninase and a cellulase fusionprotein is made by crossing-breeding the abovementioned transgenicplants one of which contains cbh1 or e1 stably integrated into thegenome and the other of which contains ckg4 or ckg5 stably integratedinto the genome using the method provided in (Zhong et al, Theor. Appl.Genet. 92: 752-761, (1996); Zhong et al, Plant Physiol. 110: 1097-1107,(1996); Zhong et al, Planta, 187: 483-489, (1992)). Transgenic plantsthat carry a low copy number of the DNA encoding the ligninase orcellulase fusion proteins are used for cross-breeding.

Briefly, transgenic maize plants that produce the ligninase fusionprotein are made as disclosed in Example 5 to make a first transgenicplant and transgenic maize plants that produce the cellulase fusionprotein are made as disclosed in Example 5 to make a second transgenicplant. The first and second transgenic plants are cross-pollinated tocreate a transgenic plant which produces both a ligninase and acellulase fusion protein. The progeny are analyzed for homozygosity andtransgenic plants that are homozygous for both the ligninase genecassette and the cellulase gene cassette are selected for furtherpropagation for seeds.

The progeny in the above crosses are used in subsequent crosses toproduce transgenic maize with both ligninase gene cassettes and one,two, or three cellulase gene cassettes or transgenic maize with two orthree cellulase gene cassettes and one ligninase gene cassette.

EXAMPLE 7

Production levels and activity of the cellulase fusion protein intransgenic maize made as in Example 5 or 6 is determined as follows.

Cellulase activity in transgenic maize is first assayed by standardmethods (Ghose. In Analytical Method B-304, rev. A, IUPAC Commission onBiotechnology. A short Report (1984)) based on the time course assay forhydrolysis of a pre-weighed sample of filter paper at pH 4.8-5.2 andtemperature of 50° C. While the filter paper assay is a standardsubstrate for cellulase activity, results using the filter paper assayare not particularly representative of the actual activity of thecellulase in plant materials containing cellulose, hemicellulose, andother sugars or sugar polymers. Therefore, a more accurate method fordetermining cellulase activity is used.

Plant material is ground and the ground material is suspended to aconcentration of up to about 5% in 0.05 M citrate buffer at pH 4.8 andincubated with shaking at 50° C. Over a 48 hour time period, samples areremoved at intervals of 0, 1, 3, 12, 24, and 48 hours. A minimal amountof sodium azide, about 0.05%, is added to the citrate buffer duringincubation to control microbial activity. For analysis by high pressureliquid chromatography (HPLC), the supernatant fraction of each sample isremoved, capped, and heated to inactivate the enzymes. The inactivatedsupernatant fraction is filtered through a syringe filter and analyzedby HPLC to measure the glucose, cellobiose, and xylose content of thesamples according to established methods (Dale et al., BiosourceTechnol. 56: 11-116 (1996)).

Cellulase activity is manifested by an increasing level of glucose,xylose and/or cellobiose levels in the supernatant fractions during the48 hour period. The control for the above assay is to treat samples fromnon-transgenic plants with varying amounts of commercially availablecellulase enzymes such as CYTOLASE 300 which is a cellulase fromGenencor, Inc. and NOVOZYME 188 which is a cellobiose from NovoLaboratories, Inc. to confirm that the ground plant material issusceptible to hydrolysis.

EXAMPLE 8

Comparison of cellulase activity in transgenic maize prepared as inExample 5 or 6 treated to enhance cellulose accessibility.

Generally, cellulose and hemicellulose in plant material are not veryaccessible to hydrolytic enzymes such as cellulase. Therefore, it ispossible that even if the cellulase fusion protein is produced in thetransgenic plants of the present invention, its cellulase activity wouldnot be measurable. Therefore, to demonstrate accessibility, samples ofthe transgenic maize plants of the present invention are treated by theammonia fiber explosion process to increase cellulose and hemicelluloseaccessibility (Dale et al., Biosource technol. 56: 11-116 (1996)).Samples treated are analyzed as in Example 3.

In previous experiments with coastal Bermuda grass, the ammonia fiberexplosion process disrupted the plant cell walls sufficiently to permitover 80% extraction of plant protein, compared with less than 30%extraction under the same conditions prior to ammonia treatment (de laRosa et al., Appl. Biochem. Biotechnol. 45/46: 483-497 (1994). Theprocess increased the hydrolytic effectiveness of the added cellulasesby at least six-fold (Dale et al., Biosource Technol. 56: 11-116(1996)). Thus, it is expected that the ammonia fiber explosion processhelps release cellulase from the transgenic maize chloroplasts and willalso increase the access of the cellulase released to the cellulose inthe plant material.

EXAMPLE 9

Production levels and activity of the ligninase fusion protein intransgenic maize made as in Example 5 or 6 can be determined as follows.

Maize leaves from the transgenic maize made as in Examples 5 or 6 areground using a pestle and mortar. Chloroplasts are isolated from leavesof transgenic plants by Ficoll (Pharmacia) gradient centrifugation andground as above.

The ground materials (leaves, grains, chloroplasts) are suspended in 50mM L-tartrate buffer (pH 4.5), mixed well by vortexing, and centrifugedfor 10 minutes at 14,000 rpm (16,000×g) at 4° C. and the supernatantfraction tested for lignin peroxidase (LIP) activity as described inTien et al., Meth. Enzymol. 161: 238-249 (1988). The LIP assay measuresthe production of veratraldehyde (as an increase in absorbance at 310nm) from veratryl alcohol (substrate) in the presence of hydrogenperoxide. Control assays are done on non-transgenic maize seeds tomeasure endogenous peroxidase activity. The assay is sensitive and isable to detect very low levels of lignin peroxidase activity, e.g.,conversion of 0.1 mmole substrate per minute per liter of test sample.

Soluble protein content is determined by the Bradford procedure(Bradford, Anal. Biochem. 72: 248-254 (1976)) using bovine serum albumen(BSA) as the standard. LIP enzyme in the extracted fluid is purified byFast Protein liquid Chromatography (FPLC) analysis using the Mono Qanion exchange system (Pharmacia) and a gradient of 0 to 1 M Na-acetateto elute the various isozymes (Yadav et al., Appl. Environ. Microbiol.61: 2560-2565 (1995); Reddy et al., FEMS Microbiol. Rev. 13: 137-152(1994)). The relative activity, yield, pH optimum, stability, and othercharacteristics of the LIP in the transgenic plant are compared to thatdetermined for the LIP isolated from the fungus. Furthermore, groundmaize seeds or leaf extracts containing the LIP is used to treat variouslignocellulosic feeds in small laboratory reactor systems and the extentof delignification can be analyzed per established procedures (Van Soestet al., Assoc. Off. Anal. Chem. J. 51: 780-785 (1968)).

Detection of ligninase mRNA is by isolating the mRNA from the transgenicplants as above, resolving the mRNA by denaturing RNA gelelectrophoresis, transferring the resolved mRNA to membranes, andprobing the membranes with ckg4 or ckg5 cDNA probes.

Western blots are performed to determine whether the LIP protein is inan active or inactive form. The total protein from the transgenic plantsis resolved by SDS-polyacrylamide gel electrophoresis and transferred tomembranes. The membranes are probed with antibodies to LIP H2 (ckg4) orLIP H10 (ckg5).

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. A transgenic plant which degrades lignins when the transgenic plantis ground to produce a plant material comprising: at least one DNAcomprising the nucleotide sequence set forth in SEQ ID NO: 11 encoding aligninase which is ckg5 which is operably linked to a nucleotidesequence encoding a signal peptide wherein the signal peptide directsthe ligninase to an organelle of the transgenic plant wherein thetransgenic plant degrades the lignins when ground to produce the plantmaterial.
 2. The transgenic plant of claim 1 wherein the DNA encodingthe ligninase is from Phanerochaete chrysosporium.
 3. The transgenicplant of claim 1 wherein the DNA encoding the ligninase is operablylinked to a leaf-specific promoter.
 4. The transgenic plant of claim 3wherein the leaf-specific promoter is a promoter for rbcS.
 5. Thetransgenic plant of claim 1 wherein the nucleotide sequence encoding thesignal peptide encodes a signal peptide of rbcS.
 6. The transgenic plantof claim 4 or 5 wherein the rbcS comprises the nucleotide sequence setforth in SEQ ID NO:1.
 7. The transgenic plant of claim 1 selected fromthe group consisting of maize, wheat, barley, rye, hops, hemp, rice,potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa,arabidopsis, coniferous tree, and deciduous tree.
 8. The transgenicplant of claim 1 wherein the DNA is stably integrated into nuclear orplastid DNA of the transgenic plant.
 9. The transgenic plant of claim 1wherein the transgenic plant further includes a DNA encoding aselectable marker operably linked to a constitutive promoter.
 10. Thetransgenic plant of claim 9 wherein the DNA encoding the selectablemarker provides the transgenic plant with resistance to an antibiotic,an herbicide, or to environmental stress.
 11. The transgenic plant ofclaim 10 wherein the DNA encoding resistance to the herbicide is a DNAencoding phosphinothricin acetyl transferase which confers resistance tothe herbicide phosphinothricin.
 12. The transgenic plant of claim 1wherein the organelle of the plant is selected from the group consistingof nucleus, microbody, endoplasmic reticulum, endosome, vacuole,mitochondria, chloroplast, or plastid.
 13. The transgenic plant of claim1 wherein the organelle of the plant is the chloroplast.